Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA)

Journal of Microbiological Methods 89 (2012) 63–70

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Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA) Mahdi Davari a, b, Anne D. van Diepeningen b,⁎, Assadollah Babai-Ahari a, Mahdi Arzanlou a, Mohammed Javad Najafzadeh c, Theo A.J. van der Lee d, G. Sybren de Hoog b, e, f, g a

Department of Plant Pathology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands Department of Parasitology and Mycology, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran d Plant Research International, Biointeractions and Plant Health, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands e Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands f Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China g Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China b c

a r t i c l e

i n f o

Article history: Received 23 December 2011 Received in revised form 24 January 2012 Accepted 25 January 2012 Available online 3 February 2012 Keywords: RCA identification Fusarium Head Blight Fusarium graminearum species complex (FGSC) Fusarium incarnatum–equiseti species complex (FIESC) Mycotoxins Gramineae

a b s t r a c t Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-α (EF-1α) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum–equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated wheat or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The International Maize and Wheat Improvement Center (CYMMIT) has appointed Fusarium Head Blight (FHB) as one of the major diseases threatening harvests and human health worldwide. This dual threat is due in part to serious crop losses in different cereals, and otherwise to the plethora of mycotoxins produced by the Fusarium species associated with FHB. To date, at least 18 species and sibling species complexes in Fusarium have been found to be able to cause FHB (Reis, 1985; Mihuta-Grimm and Foster, 1989; Bottalico and Perrone, 2002; Leonard and Bushnell, 2003). The most prevalent species causing FHB worldwide nowadays belong to the Fusarium graminearum species complex

Abbreviations: RCA, Rolling Circle Amplification; FHB, Fusarium Head Blight; FGSC, Fusarium graminearum species complex; FOSC, Fusarium oxysporum species complex; FIESC, Fusarium incarnatum and equiseti species complex; FTSC, Fusarium tricinctum species complex; EF-1α, translation elongation factor 1-α. ⁎ Corresponding author at: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel.: + 31 30 2122619; fax: + 31 30 2512097. E-mail address: [email protected] (A.D. van Diepeningen). 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2012.01.017

(FGSC), comprising at least 15 distinct lineages based on multi-locus sequence data and locally causing up to even 90–100% of the infections (Desjardins and Proctor, 2011; O'Donnell et al., 2000, 2004, 2008; Sarver et al., 2011; Starkey et al., 2007; Yang et al., 2008; Yli-Mattila et al., 2009). F. graminearum s.s. is the most important lineage worldwide (60 to more than 85% of the local infections) (e.g. Boutigny et al., 2011; Nielsen et al., 2011; Waalwijk et al., 2003; Ward et al., 2008), while F. asiaticum is prevalent in oriental Asia (40 to 90% of the local infections) (e.g. Desjardins and Proctor, 2011; Gale et al., 2002; Yang et al., 2008). Shifts in preponderance of species may occur in a field during the growth season (Landschoot et al., 2011; Xu et al., 2005). Morphologically the Fusaria are difficult to distinguish from each other, strains with similar morphology representing different biological groups composed of saprobes, endophytes and plant pathogens (Chandra et al., 2011). Closely similar species may vary in fungicide sensitivity and mycotoxin production (Nicolaisen et al., 2009). In Fusarium seven classes of mycotoxin biosynthetic genes or gene clusters have thus far been identified, including terpene cyclase, cyclic peptide synthetase, a cytochrome P450 and polyketide synthase gene clusters (Desjardins and Proctor, 2007). The species present on a single crop

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Table 1 Culture collection strains used in the current study and their reaction in the RCA assays specific for Fusarium graminearum Species complex (FGSC). Genus

Species complex

Species

CBS-number

Origin

FGSC

Fusarium

FGSC

Fusarium

Other:

acaciae-maensii (lin. 5) aethiopicum asiaticum (lin.6) asiaticum asiaticum austroamericana (lin.1) austroamericana boothii (lin.3) brasilicum cortaderiae (lin.8) cortaderiae cortaderiae gerlachii graminearum s.s (lin. 7) louisianense meridionale (lin. 2) mesoamericanum (lin. 4) nepalense nepalense nepalense ussurianum vorosii acuminatum avenaceum brachygibbosum buharicum burgessii camptoceras cerealis chlamydosporum circinatum commune culmorum flocciferum, gaditjirrii incarnatum indiyazi inflexum konzum kyushuense lacertarum langsethiae longipes nelsoni nygamai oxysporum poae poae pseudograminearum sacchari scirpi solani subglutinans torulosum tricinctum venenatum verticillioides triticina alternata malorum terreus niger flavus ochraceoroseus ochraceus terreus fumigatus pullulans sorkiniana cladosporioides herbarum ossifragi teres graminea tritici-repentis

123662 123667 110256 110257 110258 110244 110245 110270 119179 119183 123658 123659 119176 389.62 127524 110247 415.86 127503 127669 127943 123752 119177 334.75 387.62 121682 178.35 125537 544.96 589.93 635.76 100197 110089 250.52 831.85 116010 622.87 125181 716.74 119849 121807 109790 121451 120.73 119876 675.94 378.84 131025 446.67 109956 223.76 731.87 224.34 535.95 576.94 393.93 458.93 218.76 763.84 110027 Tb23 AP1 102.12 110.55 550.77 132.52 121.33 457.75 Tb20 537.72 Tb6 121281 R16 282.31 301.35 128049

Soil, Australia Triticum aestivum, Ethiopia Hordeum vulgare, Japan H. vulgare, Japan T. aesivum, China polypore, Brazil herbaceous vine, Venezuela South Africa H. vulgare, Brazil Cortaderia selloana, New Zealand H. vulgare, Brazil T. aestivum, Brazil Arundo donax, USA T. aestivum, Netherlands T. aestivum, USA Citrus × sinensis, New Caledonia Musa sapientum, Honduras Oryza sativa, Nepal O. sativa, Nepal O.sativa, Nepal Avena sativa, Russia Hungary M. sapientum, Turkey Camellia sinensis, Turkey Stone, India Gossypium, USSR Soil, Australia Leaf litter, Cuba Iris hollandica, Netherlands Cynodon lemfuensis, New Zealand Pinus taeda, Dianthus caryophyllus, Netherlands Secale cereale Triticum aestivum, Germany Heteropogon triticeus, Queensland Annato, Brazil Figs, Iran Vicia faba, Germany Sorghastrum nuttans, Kansas China Lizard Skin, India Grape vine, Syria O. sativa, Denmark Plant debris,South Africa Striga hermonthica, Sudan Globodera rostochiensis, Netherlands Secale montanum Anthoxanthum odoratum, Germany Hordeum vulgare, New South Wales Saccharum officinarum, India Triticum aestivum, Germany human toe nail, Cuba Lupinus sp. T. aestivum, Germany Triticum aestivum, Germany Triticum aestivum, Austria Zea mays, Germany Triticum aestivum, India Human keratitis, Germany Oil polluted soil, Iran Oil polluted soil, Iran – Air, Brazil Soil, Ivory Coast Phaseolus vulgaris – Soil, India Oil polluted soil, Iran Triticum sphaerococcum, Canada Oil polluted soil, Iran

+ + + + + + + + + + + + + + + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

Alternaria Alternaria Aspergillus

Aureobasidium Bipolaris Cladosporium Cladosporium Dreschlera

Oil polluted soil, Iran Japan – –

− − − − − − − − − − − − − − − − − − − − −

M. Davari et al. / Journal of Microbiological Methods 89 (2012) 63–70

65

Table 1 (continued) Genus

Species complex

Epicoccum Exophiala Geaumannomyces Penicillium Rhynchosporium Stagonospora Stemphylium Trichoderma Ulocladium

Species

CBS-number

Origin

FGSC

nigrum nigrum xenobiotica xenobiotica graminis var.tritici chrysogenum verruculosum secalis nodorum brassicicola hamatum harzianum porri

131253 120.22 Tb14 Tb3 273.36 N8 624.72 345.29 127171 124749 121.13 330.32 126314

Triticum aestivum, Iran Solanum tuberosum, Germany Oil polluted soil, Iran Oil polluted soil, Iran Tricinctum aestivum, Argentina Oil polluted soil, Iran Soil, Ukraine – – Brassica pekinensis, China Soil, Switzerland Pinus, Poland –

− −

may be able to produce a wide range of mycotoxins and even within a single species there may be variation for the toxins produced. Most FHB-related species are known to produce several mycotoxins concomitantly. In some countries checks for Fusarium infections and mycotoxin production in crops are part of daily routine with the help of advanced, often expensive techniques, but elsewhere such screening is hampered by insufficient availability of reliable and cost-effective techniques. The present study focuses on the development of a sensitive and cost-effective technique suitable for the identification of Fusarium species: Rolling Circle Amplification (RCA) of DNA is an isothermic amplification method applying so-called padlock probes, the resulting products easily being visualized on agarose gel (Fire and Xu, 1995). The 3′and 5′end strands of the probes hybridize next to one another at the target strand resulting in circularization of the molecule upon ligation. This circular molecule can then isothermically be amplified by a DNA polymerase that lacks exonuclease activity, and the resulting product in turn can be used by a second primer resulting in a cascade of amplifications. Due to the necessary precise base pairing, the padlock probes are able to detect single point mutations (Nilsson et al., 1994; 1997). The RCA technique has successfully been applied to different fungal species like human pathogens (e.g. Zhou et al., 2008; Najafzadeh et al., 2011; Sun et al., 2011), and thus far for just a few plant pathogenic fungi (Tsui et al., 2010). We developed an RCA padlock probe specific for all known species from the FGSC and tested it for specificity and potential cross reactions with other pathogenic and non-pathogenic species – Fusaria and unrelated species –, encountered in and on cereals like wheat and barley or in soil. Similarly we developed and tested specific primers for other Fusarium species; from the Fusarium incarnatum– equiseti species complex (FIESC), Fusarium oxysporum species complex (FOSC), and Fusarium tricinctum species complex (FTSC). We then used these padlock probes to characterize a set of wild isolates found in association with FHB phenomena in wheat and wild grass species in Iran. Furthermore, we aimed to prove that the FGSCprobe also is effective with extracts from wheat samples spiked with F. graminearum s.s. or F. asiaticum, without prior isolation of the pathogen. 2. Materials and methods 2.1. Fungal strains Fusarium strains and representative isolates from other genera to test RCA primers were taken from the CBS Culture Collection (Table 1). Wild-type Fusarium strains (Table 2) were collected from wheat or wild grasses heads harvested during severe FHB epidemics in 2009 and 2010 in the North and North-West of Iran using Nash & Snyder and PDA media. A first morphological characterization of Fusarium isolates was done on SNA and PDA media according to the taxonomy of Nelson et al. (1983) and Leslie and Summerell (2006). DNA-based sequence typing was done based on the Elongation Factor

− − − − − − − − −

1-alpha region (EF) compared to the FusariumID database (http:// isolate.fusariumdb.org/; Geiser et al., 2004). Wheat samples of the cultivars Lavett and Thasos were inoculated with different F. graminearum s.s. or Fusarium asiaticum isolates (Table 3), samples were milled and processed using the same procedures as the pure cultures. 2.2. DNA extraction and species identification based on EF DNA was extracted by the CTAB-based method from Möller et al. (1992). The Elongation Factor 1-alpha region was amplified by PCR with the primers EF-728 (Carbone and Kohn, 1999) and EF2 (Jacobs et al., 2004) with a modified PCR program: pre-denaturing for 5 min at 94 °C, ten cycles of 45 s at 94 °C, 45 s at 55 °C and 1.5 min at 72 °C, 30 cycles of 45 s at 94 °C, 45 s at 52 °C and 1.30 min at 72 °C, and a post-elongation step of 6 min at 72 °C and Bioline Taq polymerase (Bioline UK, London, UK) in 12.5 μl volumes. Amplicons were purified with Sephadex G-50 fine (HE Healthcare Bio-Sciences AB, Uppsala, Sweden), and subjected to direct sequencing using the ABI prism BigDye™ terminator cycle sequence kit (Applied Biosystems, Foster City, USA) and analyzed on a ABI Prism 3730XL Sequencer. Sequences were edited using SeqMan in the Lasergene software (DNASTAR, Madison, WI, USA) and for identification compared to sequences in Genbank, the Fusarium-ID database (http://isolate.fusariumdb.org/; Geiser et al., 2004) and the Fusarium MLST database (http://www.cbs. knaw.nl/fusarium/; O'Donnell et al., 2010). The top best (3–8) matches for each database coincided in species identification and were taken as the right identification. GenBank accession numbers of the partial Elongation Factor 1-alpha region of our strains are given in Table 2. 2.3. Padlock probe design For the design of the RCA padlock probes, more than 60 sequences of the elongation factor 1-α (EF-1α) region of Fusarium strains belonging to the FGSC, FIESC, FTSC, and FOSC and more than 40 strains of Fusarium spp. outside these complexes were taken from the FusariumID and MLST databases. To identify informative nucleotides polymorphisms the sequences were aligned and adjusted manually using BIONUMERICS v. 4.61 (Applied Maths, Sint-Martens-Latem, Belgium). To optimize the binding efficiency of the padlock probes to the target DNAs, the probes (Table 4) were designed with minimum secondary structure and a Tm of the 21–23 bp 5′-end close to or above the ligation temperature. The 14–16 bp 3′-end of the probe was designed with a Tm 10 to 15 º C below the average ligation temperature to increase the discriminative specificity (Najafzadeh et al., 2011). The 63 bp linker region of the padlock probes is as described by Zhou et al. (2008). 2.4. Ligation, exonucleolysis and Rolling Circle Amplification (RCA) Two microlitre of EF amplicon was mixed with 2 U pfu DNA ligase (Epicentre Biotechnologies, Madison, WI, USA) and 0.1 μmol l − 1 of a

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Table 2 Wild-type strains isolated from FHB-associated symptoms in T. aestivum and wildgrasses in the North and North-Western regions of Iran. Species classification based on EF sequences and comparison to the FusariumID database (http://isolate.fusariumdb.org/; Geiser et al., 2004), RCA-classification based on recognition by the different probes. CBS-number

Species

Origin

RCA probe

GenBank accession nr

130612 130669 130868 130889 130904 130910 130911 130914 130918 130956 130957 131005 131006 131011 131014 131017

graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s graminearum s.s. graminearum s.s graminearum s.s brachygibbosum

FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC FGSC –

JQ429353 JQ429361 JQ429360 JQ429362 JQ429363 JQ429364 JQ429365 JQ429366 JQ429367 JQ429352 JQ429354 JQ429368 JQ429369 JQ429358 JQ429359 JQ429370

131021 131024 131100 131152 131176 131178 131179 131184 131186 131188 131189 131192 131194 131199 131202 131208 131251 131252 131255 131257 131258 131261 131265 131439 131445 131574 131168(W88) 131775(Z8) 131777(Z19) 131778(Z21) 131783(Z3) 131785(Z33) 131787(Z38) T145 Z37

graminearum s.s avenaceum proliferatum reticulatum var. negundinis FIESC sp. proliferatum avenaceum acuminatum FIESC sp. torulosum equiseti tricinctum oxysporum graminearum s.s. FIESC sp. proliferatum graminearum s.s. brachygibbosum equiseti equiseti acuminatum pseudograminearum graminearum s.s. graminearum s.s. graminearum s.s. proliferatum graminearum s.s. solani FIESC sp. graminearum s.s. graminearum s.s. proliferatum equiseti graminearum s.s. commune

T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran Hordeum murinum, Iran T. aestivum, Iran Alopecurus mycosuroides, Iran A. mycosuroides, Iran Aegilops triuncialis, Iran Paspalum paspaloides, Iran A. triuncialis, Iran Setaria gluaca, Iran Wild grass, Iran A. triuncialis, Iran A. triuncialis, Iran Wild grass, Iran Wild grass, Iran Wild grass, Iran Dactylis glomereta, Iran Arrhenatherum elatius, Iran Leucopoa sclerophylla, Iran Bromus inermis, Iran Cynodon dactylon, Iran T. aestivum, Iran T. aestivum, Iran L. sclerophylla, Iran L. sclerophylla, Iran Lolium sp., Iran Hordeum vulgare, Iran T. aestivum, Iran Echinocloa crus-galii, Iran T. aestivum, Iran T. aestivum, Iran E. crus-galii, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran T. aestivum, Iran

FGSC FATAV – – FIESC – FATAV FATAV FIESC – FIESC FATAV FOXY FGSC FIESC – FGSC FIESC FIESC FATAV – FGSC FGSC FGSC – FGSC – FIESC FGSC FGSC – FIESC FGSC –

JQ429371 JQ429372 JQ429350 JQ429356 JQ429357 JQ429373 JQ429374 JQ429375 JQ429351 JQ429376 JQ429377 JQ429378 JQ429355 JQ429330 JQ429331 JQ429332 JQ429333 JQ429334 JQ429335 JQ429336 JQ429337 JQ429338 JQ429339 JQ429329 JQ429340 JQ429341 JQ429343 JQ429344 JQ429345 JQ429328 JQ429346 JQ429347 JQ429349 JQ429342 JQ429348

Table 3 Wheat samples inoculated with different chemotypes of Fusarium graminearum s.s or Fusarium asiaticum tested positive with the FGSC probe. Cultivar

Inoculates with

Lavett Lavett Lavett Lavett Lavett Lavett Thasos Thasos Thasos Thasos Thasos Thasos Control

Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium Fusarium None

graminearum graminearum graminearum asiaticum asiaticum asiaticum graminearum graminearum graminearum asiaticum asiaticum asiaticum

s.s. s.s. s.s.

s.s. s.s. s.s.

Strain

Mycotoxin

FGSC probe

16D1 68D2 31F1 Bfb0982-1 CH024b Bfb0082-1 16D1 68D2 31F1 Bfb0982-1 CH024b Bfb0082-1 –

NIV 15ADON 3ADON NIV 15ADON 3ADON NIV 15ADON 3ADON NIV 15ADON 3ADON –

+ + + + + + + + + + + + −

padlock probe in 20 mmol l − 1 Tris–HCl (pH7.5), 20 mmol l − 1 KCl, 10 mmol l − 1 MgCL2, 0.1% Igepal, 0.01 mmol l − 1 rATP and 1 mmol l − 1 DTT in a total reaction volume of 10 μl. The ligation reaction was performed as described by Wang et al. (2005) by one denaturation cycle for 5 min at 94 °C, followed by five cycles of 30 s. at 94 °C and 4 min ligation at 63 °C. In the initial experiments an exonucleolysis step was performed to remove unligated padlock probes by addition of 10 U each of both exonucleases I and III (New England Biolabs, Hitchin, UK) to the ligation mixture in a final volume of 20 μl and an incubation of 30 min at 37 °C, followed by 3 min at 94 °C to inactivate the endonucleases. However, this step proved not necessary and only reduces the chances of ligation-independent amplification events (Sun et al., 2011). For the RCA, 4 μl of the ligation product was used with 8 U Bst DNA polymerase (New England Biolabs, Ipswich, MA, USA), 400 μmol l − 1

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Fig. 1. Gel electrophoresis of RCA reactions with the Fusarium graminearum species complex specific probe. Lanes 1–15: positive reactions with the FGSC probe; 1 — Fusarium acaciae-maensii (CBS 123662), 2 — F. aethiopicum (CBS 123667), 3 — F. asiaticum (CBS 110256), 4 — F. austroamericana (CBS 110244), 5 — F. boothii (CBS 110270), 6 — F. brasilicum (CBS 119179), 7 — F. cortaderiae (CBS 119183), 8 — F. gerlachii (CBS 119176), 9 — F. graminearum s.s. (CBS 389.62), 10 — F. meridionale (CBS 110247), 11 — F. mesoamericanum (CBS 415.86), 12 — F. nepalense (CBS 127503), 13 — F. ussurianum (CBS 123752), 14 — F. vorosii (CBS 119177), 15- F. louisianense (CBS 127524); lanes 16–25: negative reaction with the FGSC probe; 16 — F. brachygibbosum (CBS 131017), 17 — F. culmorum (CBS 250.52), 18 — F. equiseti (CBS 131189) 19 — Penicillium verruculosum (CBS 642.72), 20 — F. proliferatum (CBS 131208), 21 — Trichoderma hamatum (CBS 121.13), 22 — F. oxysporum (CBS 378.84), 23 — Bipolaris sorkiniana (CBS 537.72), 24 — Aspergillus flavus (CBS 110.55), 25 — Control, M = 100 bp molecular weight marker.

dNTP mix, 10 pmol of each of the RCA primers (Table 2) in a final volume of 50 μl. Probe signals were amplified by incubating 60 min at 65 °C and accumulated dsDNA products were visualized on 1% agarose gel, stained with GelRed™ (Biotium, Hayward, CA, USA). Positive reactions showed a ladder-like pattern, while negative reactions showed a clean background [when the exonuclease step is omitted some faint signal may be visible in gel electrophoresis (Sun et al., 2011)]. For direct visual detection of DNA in the RCA detection mixtures 1.0 μl of a 10-fold diluted original SYBR Green I (Cambrex Bio Science, Workingham, U.K.) can be added to the reaction tubes and visualized on a UV transilluminator (Vilber Lourmat, Marne-laVallée, France). 3. Results A positive RCA signal was clearly visualized with gel electrophoresis and any fluorescent dye binding to DNA as a ladder of fragments increasing in size, comprising the monomer and multimer repeats of the amplified product formed by single and multiple copying of the circularized padlock probe (Fig. 1). Using the two padlock-probe specific primers in the RCA reaction an increase was observed in the resulting signal that could even be visualized by simply adding fluorescent dyes directly to the reaction mixture and putting these on a UV transilluminator, without having to put the mixtures on gel (Fig. 2). For many fungi, sequence data of the nuclear ribosomal internal transcribed spacer (ITS) region or of domains D1 and D2 of the nuclear large-subunit ribosomal DNA (LSU) yield enough data to distinguish entities down to the species level. However, in Fusarium these loci are too conserved to distinguish between closely related species. Therefore introns of nuclear genes like the Elongation Factor 1-alpha region (EF) are used for correct identification (O'Donnell et al., 2010).

We used the EF-fragment in strains phenotypically characterized to belong to the F. graminearum species complex and of related Fusarium species to design a padlock probe specific for the FGSC. This probe was tested on a set of known FGSC strains, on other Fusarium species, and on species from other genera that can be encountered on cereals, other plants and in soils. Our FGSC probe proved to be specific for the identification of the currently known lineages within the FGSC, without cross-reactions to other species. Isolates from all over the world were readily identified (Table 1). In addition to members of the F. graminearum species complex, also other Fusaria are regularly found in association with FHB. For three of these complexes, FIESC, FTSC, and FOSC, RCA primers were also developed. These padlock probes were tested for specificity to target- and non-target species (Fig. 3). The FOXY probe identified different F. oxysporum sensu stricto strains and none of the other tester species in FOSC or other Fusarium complexes. The FIESC probe identified all tested species from the FIESC including F. equiseti, F. incarnatum, F. lacertarum, F. sciripi, but none of the species of other complexes. The FATAV probe specifically identified three important species belonging to the FTSC: F. acuminatum, F. tricinctum and F. avenaceum (hence FATAV), but not two remaining species of this complex, F. flocciferum and F. negundis (Table 5). To further validate our RCA padlock probes for correct species identification we applied them to characterize a set of strains isolated from recent epidemics of FHB; these strains had also been identified with EF sequencing. The probes correctly recognized 100% of the species, while no cross-reactivity was observed (Table 2). In addition, milled wheat samples from two cultivars contaminated with either F. graminearum s.s. or F. asiaticum were used for RCA analysis. All contaminated samples yielded a positive response in our tests without false negatives or false positives (Table 3). 4. Discussion

Fig. 2. Visual detection of DNA in RCA detection mixtures by adding SYBR Green I under UV light. From left to right the used strains are 1 — F. graminearum (CBS 130657), 2 — F. boothii (CBS 110270), 3 — F. graminearum s.s. (CBS 389.62), 4 — F. austroamericana (CBS 110245) (positive signal) and 5 — Trichoderma hamatum (CBS 121.13), 6 — Penicillium verruculosum (CBS 642.72), 7 — Bipolaris sorkiniana (CBS 537.72), and 8 — Fusarium proliferatum (CBS131208) (negative signal) with the FGSC probe.

Fusarium Head Blight (FHB) is a disease causing severe crop losses and mycotoxin production in cereals all over the world. The disease can be caused by different Fusarium species and species complexes, of which the F. graminearum species complex (FGSC) seems to comprise the primary etiological agents world-wide at the moment. In the present study, we evaluated an identification method based on Rolling Circle Amplification (RCA) enabling rapid and specific detection of single nucleotide differences. We developed four padlock probes on the basis of the Elongation Factor 1-alpha (EF) region to identify species from either FGSC, FOSC, FIESC or FTSC; species all frequently encountered on cultivated and wild gramineous hosts and capable of producing a wide range of mycotoxins. The probes allowed specific amplification of the target groups, while no cross-reactivity was observed, neither with other Fusarium species/ complexes, nor

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Fig. 3. Gel representation of specificity of Rolling Circle Amplification probes. Amplification of probe signals was seen only with matched template–probe mixtures. The used RCA probes are given at the top of the gel: FGSC, Fusarium graminearum complex species; FIESC, Fusarium incarnatum–equiseti complex species; FATAV, Fusarium acuminatum/tricinctum/ avenaceum species and FOXY, Fusarium oxysporum; lanes 1, 5, 9, and 13, F. graminearum (CBS 389.62); lanes 2, 6, 10 and 14, F. equiseti (CBS 131015); lanes 3,7,11 and 15, F. acuminatum (CBS 131192) and lanes 4, 8,12 and 16, F. oxysporum (CBS 378.84). M = 100 bp molecular weight marker.

with unrelated fungal species. The amplification product can be visualized by agarose gel electrophoresis, but can also be visualized in gelfree systems using fluorescence staining of amplified product by SYBR Green in combination with a UV trans illuminator. The technique can be used for species identification, but also shows potential for the detection of FGSC in wheat samples. Thus, the RCA method provides a powerful tool for rapid species resp. species complex identification of Fusarium species associated with Head Blight in wheat and wild grasses. Sequencing of the internal transcribed space (ITS) is the gold standard for species identification, but for many Fusarium species elongation factor 1-α (EF-1α) proves to be better for specific identification, and also provides more phylogenetic information. Where ITS sequences may vary even within a single nucleus due to the presence of non-orthologous copies, or not to vary at all (O'Donnell and Cigelnik, 1997; O'Donnell et al., 1998, 2010). EF sequences have been used to show correlations between phylogeny and toxigenic potential of Fusarium species, such as the ability to produce zearalenone and type B trichothecenes in FGSC and moniliformin production in FTSC (Kristensen et al., 2005). Our probes were not based on mycotoxin genes since this would only tell us which toxins are potentially produced, whereas with EF sequences much larger groups of probably toxigenic species were identified. The Luminex assay developed for the detection and identification of species of the FGSC and close relatives was also based on differences in EF-1α and on other coding genes (Ward et al., 2008). Although, a Luminex assay has the benefit of detecting many different species in a single run it requires sensitive and expensive equipment and reagents. RCA on the other hand does not require expensive instrumentation and is therefore more suitable for local, point of care measurements. We demonstrate that using basic equipment it is also possible to achieve single base-pair resolution. Samples that are positive for one of the species complexes may be further tested for

full species identification. With the use of RCA primers such as the ones we developed and based on a single polymorphic gene but with specificity for different species/species complexes, it may be decided to use a single PCR sample to further identify species identities present in the sample. To this aim, parallel RCA reactions with different primers for each species/ species complex are performed. Alternatively, padlock probes differing in size leading to fragment ladders with different interval recognizable on gel electrophoresis can be applied. However, in most cases this may not be needed. Another low-cost alternative to RCA may be loop-mediated isothermal amplification (LAMP). This method uses a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene (Niessen and Vogel, 2010). Niessen and Vogel (2010) based their identification of species from the FGSC on the galactose oxidase precursor (gaoA), a less often studied gene in this genus. Amplification of DNA during the reaction was indirectly detected in situ by using calcein fluorescence as a marker without the necessity of time-consuming electrophoretic analysis. However, Nilsson et al. (2002) also described an RCA identification method in Real-time and the present paper showed that direct visible identification is also possible. Najafzadeh et al. (2011) compared the RCA and LAMP detection for human pathogenic Fonsecaea species and found that LAMP was highly sensitive, but RCA proved to be more specific. The development of RCA probes to distinguish single species or groups of species relies on the presence of sufficient sequence data and useful species-specific polymorphisms in genes of correctly identified species. While in GenBank many sequences can be found, correct identification of the species cannot always be guaranteed. For Fusarium two overlapping databases exist that contain multi-locus sequence data of well-classified strains: the FusariumID database and the Fusarium MLST database, containing e.g. a wealth of data on EF1α.

Table 4 The Rolling Circle Amplification padlock probes and padlock probe-specific primers used in this study. Oligonucleotidesa

Sequencesb

RCA1 RCA2 FGSC FOXY FIESC FATAV

5′-ATGGGCACCGAAGAAGCA-3′ 5′-CGCGCAGACACGATA-3′ 5′-P-GTGACAACATACCAATGACGGTGgatcaTGCTTCTTCGGTGCCCATtacgaggtgcggatagctacCGCGCAGACACGATAgtctaTGATGACAGCAGTG-3′ 5′-P-GAGTACTCACAGTGGTCGACTTGgatcaTGCTTCTTCGGTGCCCATtacgaggtgcggatagctacCGCGCAGACACGATAgtctaCTCATTGTCGAGGA-3′ 5′-P-GCAATYTTGTCAGCARATRTDYAgatcaTGCTTCTTCGGTGCCCATtacgaggtgcggatagctacCGCGCAGACACGATAgtctaAGTGACCGGTCTAT-3′ 5′-P-GCGCGTAATCGAAGGGATATTgatcaTGCTTCTTCGGTGCCCATtacgaggtgcggatagctacCGCGCAGACACGATAgtctaGGGAATCGATGGGAGC-3′

a RCA, rolling circle amplification; FGSC, Fusarium graminearum species complex; FOXY, Fusarium oxysporum; FIESC, Fusarium incarnatum–equiseti species complex; FATAV, Fusarium avenaceum, F. acuminatum and F. tricinctum. b P at the 5′end of the probe indicates 5′-phosporilation, the underlined capital sequences are the species (group) specific binding arms of the padlock probes, in small letters the linker regions with therein in bold capital letters the binding sites for the padlock probe-specific primers.

M. Davari et al. / Journal of Microbiological Methods 89 (2012) 63–70

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Table 5 The reaction of different fungal strains in RCA assays with probes specific for the Fusarium oxysporum (FOXY), the Fusarium incarnatum-equiseti Species complex (FIESC) and F. acuminatum, F. avenaceum and F. tricinctum species complex (FATAV). Probe

+ positive signal species

CBS-numbers

− negative signal Species

CBS-numbers

FOXY

F.oxysporum

378.84; 579.93; 131194; 141.95

FIESC

F. equiseti F. incarnatum F. lacertarum Fusarium sp. FIESC F. acuminatum F. avenaceum F. tricinctum

131015; 131189; 131255; 131257 622.87; 131198 109796 131009; 131072; 131084; 131090; 131098; 131155; 131174; Z38 131258; 131192 131024, 131179 393.93; 131184; 131193

F. acuminatum F. commune F. graminearum s.s. F. inflexum F. proliferatum F. redolens F. acuminatum F. graminearum s.s. F. oxysporum F. proliferatum F. acacia-mearnsii F. aethiopicum F. austroamericanum F. brasilicum F. buharicum F. culmorum F. flocciferum F. gaditjirii F. graminearum s.s. FIESC sp. F. negundis F. oxysporum F. poae F. proliferatum F. pseudograminearum F. ussurianum F. vorosii

131258 110089 130612, Z34 716.74 131208 131187 131258 130612 131194 131208 123662 123667 110245 119179 178.35 250.52 831.85

FATAV

With the availability of well-administrated Fusarium sequence databases containing data on multiple genes, additional RCA primers can be designed for specific species or groups of species. The RCA assays successfully amplified DNA of the target fungi, while no cross reactivity with non-target DNA was observed. RCA does not require highly specialized equipment, while it is relatively easy expanded to multiple or routine identifications. In conclusion, the technique has a good potential for rapid screening of (potentially toxigenic) species in cereal crops.

Acknowledgments First author Mahdi Davari was financially supported by the Iranian Ministry of Science, Research and Technology, University of Tabriz. We thank Mr. Kasper Luijsterburg for his help with the photography, Mr. Bert Gerrits van den Ende and Mr. Arne Noordegraaf for their practical help with the DNA work and Dr. Benjamin Stielow, Dr. Karolina Dukik and Dr. Hugo Madrid for providing some of the DNA samples.

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